Combination therapy for treating hcv infection

ABSTRACT

The present invention relates to therapeutic combinations comprising (a) Compound (1), or a pharmaceutically acceptable salt thereof, as herein described, (b) an interferon alfa and (c) ribavirin. Compound (1) is a selective and potent inhibitor of the HCV NS3 serine protease. The present invention also relates to methods of using such therapeutic combinations for treating HCV infection or alleviating one or more symptoms thereof in a patient.

This application claims the benefit of prior U.S. ProvisionalApplications 61/097,753 filed Sep. 17, 2008, 61/109,033 filed Oct. 28,2008 and 61/171,935 filed Apr. 23, 2009.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to therapeutic combinations comprisingCompound (1) as herein described, an interferon alfa and ribavirin. Thepresent invention also relates to methods of using such therapeuticcombinations for treating HCV infection or alleviating one or moresymptoms thereof in a patient. The present invention also provides kitscomprising the therapeutic combinations of the present invention.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infection is a global human health problem withapproximately 150,000 new reported cases each year in the United Statesalone. HCV is a single stranded RNA virus, which is the etiologicalagent identified in most cases of non-A, non-B post-transfusion andpost-transplant hepatitis and is a common cause of acute sporadichepatitis. It is estimated that more than 50% of patients infected withHCV become chronically infected and 20% of those develop cirrhosis ofthe liver within 20 years.

Several types of interferons, in particular, alfa-interferons areapproved for the treatment of chronic HCV, e.g., interferon-alfa-2a(ROFERON®-A), interferon-alfa-2b (INTRON®-A), consensus interferon(INFERGEN®), as well as pegylated forms of these and other interferonslike pegylated interferon alfa-2a (PEGASYS®) and pegylated interferonalfa-2b (PEG-INTRON®). Most patients are unresponsive to interferon-alfatreatment, however, and among the responders, there is a high recurrencerate within 6 months after cessation of treatment (Liang et al., J. Med.Virol. 40:69, 1993).

Ribavirin, a guanosine analog with broad spectrum activity against manyRNA and DNA viruses, has been shown in clinical trials to be effectiveagainst chronic HCV infection when used in combination withinterferon-alfas (see, e.g., Poynard et al., Lancet 352:1426-1432, 1998;Reichard et al., Lancet 351:83-87, 1998), and this combination therapyhas been approved for the treatment of HCV: REBETRON® (interferonalfa-2b plus ribavirin, Schering-Plough); PEGASYS®RBV® (pegylatedinterferon alfa-2a plus ribavirin combination therapy, Roche); see alsoManns et al, Lancet 358:958-965 (2001) and Fried et al., 2002, N. Engl.J. Med. 347:975-982. However, even with this combination therapy thevirologic response rate is still at or below 50%.

Furthermore, there are significant side-effects typically associatedwith such therapies. Ribavirin suffers from disadvantages that includeteratogenic activity, interference with sperm development, haemolysis,fatigue, headache, insomnia, nausea and/or anorexia. Interferon alfa,with or without ribavirin, is associated with many side effects. Duringtreatment, patients must be monitored carefully for flu-like symptoms,depression, rashes and abnormal blood counts. Patients treated withinterferon alfa-2b plus ribavirin should not have complications ofserious liver dysfunction and such subjects are only considered fortreatment of hepatitis C in carefully monitored studies.

Certain interferon-containing combination therapies for treating HCVinfection are also disclosed in the following U.S. Patent ApplicationPublications: US 2005/0112093; US 2005/0129659; and US 2008/0138316.

The following Compound (1):

is known as a selective and potent inhibitor of the HCV NS3 serineprotease and useful in the treatment of HCV infection. Compound (1)falls within the scope of the acyclic peptide series of HCV inhibitorsdisclosed in U.S. Pat. Nos. 6,323,180, 7,514,557 and 7,585,845. Compound(1) is disclosed specifically as Compound #1055 in U.S. Pat. No.7,585,845, and as Compound #1008 in U.S. Pat. No. 7,514,557. Compound(1), and pharmaceutical formulations thereof, can be prepared accordingto the general procedures found in the above-cited references, all ofwhich are herein incorporated by reference in their entirety. Preferredforms of Compound (1) include the crystalline forms, in particular thecrystalline sodium salt form, which can be prepared as described in theexamples section herein.

Compound (1) may also be known by the following alternate depiction ofits chemical structure, which is equivalent to the above-describedstructure:

wherein B is

L⁰ is MeO-; L¹ is Br; and R² is

Although Compound (1) has been found generally effective in thereduction of viral load and the treatment of HCV infection, a certainamount of viral resistance with resulting viral rebound has been seen.For example, we have observed with Compound (1) given once-daily totreatment-naïve patients as monotherapy for 14 days a strong and veryrapid antiviral affect followed by a certain amount of resistanceformation after 5-6 days.

Therefore, there is a continuing need in the field for alternativetherapies for the treatment and prevention of HCV infection.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of treating HCV infection oralleviating one or more symptoms thereof in a patient comprising thestep of administering to the patient a therapeutic combinationcomprising a Compound (1) as defined herein, or a pharmaceuticallyacceptable salt thereof, together with an interferon alfa and ribavirin,as defined herein. The three actives of the combination can beadministered simultaneously or separately, as part of a regimen.

The present invention further provides for a kit comprising a firstpharmaceutical composition comprising a Compound (1) or apharmaceutically acceptable salt thereof; a second pharmaceuticalcomposition comprising an interferon alfa; and a third pharmaceuticalcomposition comprising ribavirin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the average change in HCV viral load in four patient dosegroups for treatment-naïve patients with chronic HCV genotype-1infection treated with Compound (1) sodium salt as monotherapy for 14days, followed by combination therapy with Compound (1) sodium salt,pegylated interferon alfa-2a and ribavirin for an additional 14 days.

FIG. 2 depicts the average change in HCV viral load in three patientdose groups for treatment-experienced patients with chronic HCVgenotype-1 infection treated with Compound (1) sodium salt, pegylatedinterferon alfa-2a and ribavirin as combination therapy for 28 days.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Compound (1)” is as defined above.

“Interferon” means a member of a family of highly homologousspecies-specific proteins that inhibit viral replication and cellularproliferation and modulate immune response. Human interferons aregrouped into three classes based on their cellular origin andantigenicity: α-interferon (leukocytes), β-interferon (fibroblasts) andγ-interferon (B cells). Recombinant forms of each group have beendeveloped and are commercially available. Subtypes in each group arebased on antigenic/structural characteristics. At least 24 interferonalfas (grouped into subtypes A through H) having distinct amino acidsequences have been identified by isolating and sequencing DNA encodingthese peptides. The terms “α-interferon”, “alfa-interferon” and“interferon alfa” are used interchangeably in this application todescribe members of this group. Both naturally occurring and recombinantalfa-interferons, including consensus interferon, may be used in thepractice of the invention.

Suitable interferon-alfas for the present invention include, but are notlimited to, recombinant interferon alfa-2b such as INTRON®-A interferonand VIRAFERON®; recombinant interferon alfa-2a such as ROFERON®interferon; recombinant interferon alfa-2c such as BEROFOR® alfa 2interferon; interferon alfa-n1, a purified blend of natural alfainterferons such as SUMIFERON® or WELLFERON® interferon alfa-n1 (INS);or a consensus alfa interferon such as those described in U.S. Pat. Nos.4,897,471 and 4,695,623; or interferon alfa-n3, a mixture of naturalalfa interferons such as ALFERON®. The use of interferon alfa-2a or alfa2b is preferred. The manufacture of interferon alfa 2b is described inU.S. Pat. No. 4,530,901.

The term “interferon alfa” is further intended to include those“pegylated” analogs meaning polyethylene glycol modified conjugates ofinterferon alfa, preferably interferon alfa-2a and -2b. The preferredpolyethylene-glycol-interferon alfa-2b conjugate is PEG₁₂₀₀₀-interferonalfa 2b. The term “PEG₁₂₀₀₀-IFN alfa” as used herein means conjugatessuch as are prepared according to the methods of InternationalApplication No. WO 95/13090 and containing urethane linkages between theinterferon alfa-2a or -2b amino groups and polyethylene glycol having anaverage molecular weight of 12000.

The preferred PEG₁₂₀₀₀-interferon alfa-2b is prepared by attaching a PEGpolymer to the epsilon amino group of a lysine residue in the IFNalfa-2b molecule. A single PEG₁₂₀₀₀ molecule is conjugated to free aminogroups on an IFN alfa-2b molecule via a urethane linkage. This conjugateis characterized by the molecular weight of PEG₁₂₀₀₀ attached. ThePEG₁₂₀₀₀-IFN alfa-2b conjugate is formulated as a lyophilized powder forinjection. The objective of conjugation of IFN alfa with PEG is toimprove the delivery of the protein by significantly prolonging itsplasma half-life, and thereby provide protracted activity of IFN alfa.

Especially preferred conjugates of interferon alfa that may be used inthe present invention are pegylated alfa-interferons, e.g., pegylatedinterferon alfa-2a, pegylated interferon alfa-2b, pegylated consensusinterferon or pegylated purified interferon alfa product. Pegylatedinterferon alfa-2a is described, e.g., in European Patent No. EP 0 593868 and commercially-available, e.g., under the tradename PEGASYS®(Hoffmann-La Roche). Pegylated interferon alfa-2b is described, e.g., inU.S. Pat. No. 5,908,621 and WO 98/48840 and commercially-available,e.g., under the tradename PEG-INTRON® A (Schering Plough). Pegylatedconsensus interferon is described in WO 96/11953. The preferredpegylated alfa interferons are pegylated interferon alfa-2a andpegylated interferon alfa-2b. Also preferred is pegylated consensusinterferon.

The term “interferon alfa” further includes other interferon alfaconjugates that can be prepared by coupling an interferon alfa to awater-soluble polymer. A non-limiting list of such polymers includesother polyalkylene oxide homopolymers such as polyethylene glycol (PEG),polypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof. As an alternative to polyalkyleneoxide-based polymers, effectively non-antigenic materials such asdextran, polyvinylpyrrolidones, polyacrylamides, polyvinyl alcohols,carbohydrate-based polymers and the like can be used. Such interferonalfa-polymer conjugates are described in U.S. Pat. No. 4,766,106, U.S.Pat. No. 4,917,888, European Patent Application No. 0 236 987, EuropeanPatent Application Nos. 0510 356, 0 593 868 and 0 809 996 (pegylatedinterferon alfa-2a) and International Publication No. WO 95/13090.

The term “interferon alfa” further includes fusion proteins of aninterferon alfa, for example fusion proteins of interferon-α-2a,interferon-α-2b, consensus interferon or purified interferon-α product,each of which is fused with another protein. Certain preferred fusionproteins comprise an interferon (e.g., interferon-α-2b) and an albuminas described in U.S. Pat. No. 6,972,322 and international publicationsWO2005/003296 and WO2005/077042. A preferred interferon conjugated to ahuman albumin is ALBUFERON® which is a longer-acting form of interferonalfa created using albumin fusion technology. ALBUFERON® results fromthe genetic fusion of human albumin and interferon alfa. Also includedare consensus interferons, such as INFERGEN®.

The term “pharmaceutically acceptable salt” means a salt of a Compoundof formula (1) which is, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, generally water oroil-soluble or dispersible, and effective for their intended use.

The term includes pharmaceutically-acceptable acid addition salts andpharmaceutically-acceptable base addition salts. Lists of suitable saltsare found in, e.g., S. M. Birge et al., J. Pharm. Sci., 1977, 66, pp.1-19.

The term “pharmaceutically-acceptable acid addition salt” means thosesalts which retain the biological effectiveness and properties of thefree bases and which are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, and thelike, and organic acids such as acetic acid, trifluoroacetic acid,adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoicacid, butyric acid, camphoric acid, camphorsulfonic acid, cinnamic acid,citric acid, digluconic acid, ethanesulfonic acid, glutamic acid,glycolic acid, glycerophosphoric acid, hemisulfic acid, hexanoic acid,formic acid, fumaric acid, 2-hydroxyethane-sulfonic acid (isethionicacid), lactic acid, hydroxymaleic acid, malic acid, malonic acid,mandelic acid, mesitylenesulfonic acid, methanesulfonic acid,naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid,oxalic acid, pamoic acid, pectinic acid, phenylacetic acid,3-phenylpropionic acid, pivalic acid, propionic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaricacid, p-toluenesulfonic acid, undecanoic acid, and the like.

The term “pharmaceutically-acceptable base addition salt” means thosesalts which retain the biological effectiveness and properties of thefree acids and which are not biologically or otherwise undesirable,formed with inorganic bases such as ammonia or hydroxide, carbonate, orbicarbonate of ammonium or a metal cation such as sodium, potassium,lithium, calcium, magnesium, iron, zinc, copper, manganese, aluminum,and the like. Particularly preferred are the ammonium, potassium,sodium, calcium, and magnesium salts. Salts derived frompharmaceutically-acceptable organic nontoxic bases include salts ofprimary, secondary, and tertiary amines, quaternary amine compounds,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion-exchange resins, such as methylamine,dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine,isopropylamine, tripropylamine, tributylamine, ethanolamine,diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine,choline, betaine, ethylenediamine, glucosamine, methylglucamine,theobromine, purines, piperazine, piperidine, N-ethylpiperidine,tetramethylammonium compounds, tetraethylammonium compounds, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, N,N′-dibenzylethylenediamine, polyamine resins, and thelike. Particularly preferred organic nontoxic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline,and caffeine.

“Ribavirin” refers to1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available from ICNPharmaceuticals, Inc., Costa Mesa, Calif. and is described in the MerckIndex, compound No. 8199, Eleventh Edition. Its manufacture andformulation is described in U.S. Pat. No. 4,211,771. Preferred marketedribavirin products include REBETOL® and COPEGUS®. The term furtherincludes derivatives or analogs thereof, such as those described in U.S.Pat. Nos. 6,063,772, 6,403,564 and 6,277,830. For example, derivativesor analogs include modified ribavirins such as 5′-amino esters, ICNPharmaceutical's L-enantiomer of ribavirin (ICN 17261), 2′-deoxyderivatives of ribavirin and 3-carboxamidine derivatives of ribavirin,viramidine (previously known as ribamidine) and the like.

The term “therapeutic combination” as used herein means a combination ofone or more active drug substances, i.e., compounds having a therapeuticutility. Typically, each such compound in the therapeutic combinationsof the present invention will be present in a pharmaceutical compositioncomprising that compound and a pharmaceutically acceptable carrier. Thecompounds in a therapeutic combination of the present invention may beadministered simultaneously or separately, as part of a regimen.

EMBODIMENTS OF THE INVENTION

According to a general embodiment, the present invention provides for amethod of treating HCV infection or alleviating one or more symptomsthereof in a patient comprising the step of administering to the patienta therapeutic combination comprising a Compound (1) as defined herein,or a pharmaceutically acceptable salt thereof, together with aninterferon alfa and ribavirin. In another embodiment, the presentinvention teaches the use of a Compound (1) as defined herein, or apharmaceutically acceptable salt thereof, an interferon alfa, andribavirin for the preparation of a pharmaceutical kit to treat ahepatitis C viral (HCV) infection or alleviating one or more symptomsthereof in a patient. Although this combination therapy is expected tobe effective against all HCV genotypes, it has been demonstrated to beparticularly effective in treating HCV genotype 1 infection, includingsubgenotypes 1a and 1b.

The patient population to be treated with the combination therapy of thepresent invention can be further classified into “treatment-naïve”patients, i.e., those patient who have not received any prior treatmentfor HCV infection and “treatment experienced” patients, i.e, thosepatients who have undergone prior treatment for HCV. Either of theseclasses of patients may be treated with the combination therapy of thepresent invention. A particular class of patients that are preferablytreated are those treatment experienced patients that have undergoneprior interferon plus ribavirin therapy but are non-responsive to saidtherapy (herein “non-responders”). Such non-responders include threedistinct groups of patients: (1) those who experienced <1×log₁₀ maximumreduction in HCV RNA levels during treatment with interferon plusribavirin (“null responders”), (2) those who experienced ≧1×log₁₀maximum reduction in HCV RNA levels during treatment with interferonplus ribavirin but never achieve HCV RNA levels below level of detection(“partial responders”), and (3) those who achieved a virologic responsewith and during interferon plus ribavirin therapy but had a viral loadrebound either during treatment (other than due to patientnon-compliance) or after treatment has completed (“relapser”). As willbe detailed below, particularly surprising results have been obtainedwith the treatment of certain non-responder patients using thecombination therapy regimen of the present invention.

According to an alternative embodiment, the present invention provides amethod of reducing HCV-RNA levels in a patient in need thereof,comprising the step of administering to said patient a therapeuticcombination according to the present invention. Preferably, the methodof the present invention reduces the HCV-RNA levels in a patient to aless than detectable level. The terms “less than detectable level” and“below level of detection” are used interchangeably herein and have thesame meaning as being less than a detectable level of HCV RNA. Adetectable level of HCV RNA as used in the present invention means atleast 50 International Units (IU) per ml of serum of a patient asmeasured by quantitative, multi-cycle reverse transcriptase PCRmethodology according to the WHO international standard (Saladanha J,Lelie N and Heath A, Establishment of the first international standardfor nucleic acid amplification technology (NAT) assays for HCV RNA. WHOCollaborative Study Group. Vox Sang 76:149-158, 1999). Such methods arewell known in the art. In a preferred embodiment, the method of thepresent invention reduces the HCV-RNA levels in a patient to less than25 IU per ml of serum, even more preferably to less than 10 IU per ml ofserum.

The usual duration of the treatment for standard interferon plusribavirin therapy is at least 48 weeks for HCV genotype 1 infection, andat least 24 weeks for HCV genotypes 2 and 3. However, with the additionof Compound (1), or a pharmaceutically acceptable salt thereof, in thetriple combination therapy of the present invention, it may be possibleto have a much shorter duration of treatment. With the triplecombination therapy of the present invention the contemplated durationsof treatment include at least 4 weeks, preferably at least 12 weeks,e.g., from about 12 weeks to about 24 weeks, although treatment up toand even beyond 48 weeks is possible as well. Thus, further embodimentsinclude treatment for at least 24 weeks and for at least 48 weeks. Thetime period for different HCV genotypes, e.g. HCV genotypes 2, 3 or 4 isexpected to be similar. Also contemplated is an initial treatmentregimen with the triple combination therapy of the present invention,followed by a continuation of only the interferon plus ribavirin doublecombination therapy. Thus, possible scenarios for the initial triple andthen double combination therapy include, for example: (1) 4 weeks of thetriple combination therapy, followed by 20 to 44 weeks of the interferonplus ribavirin only therapy; (2) 12 weeks of the triple combinationtherapy, followed by 12 to 36 weeks of the interferon plus ribavirinonly therapy; and (3) 24 weeks of the triple combination therapy,followed by 12 to 24 weeks of the interferon plus ribavirin onlytherapy.

The first component of the therapeutic combination, namely, Compound (1)or a pharmaceutically acceptable salt thereof is comprised in acomposition. Such a composition comprises Compound (1), or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable adjuvant or carrier. Typical pharmaceutical compositions thatmay be used for Compound (1), or a pharmaceutically acceptable saltthereof, are as described in U.S. Pat. No. 7,514,557.

In general, the Compound (1) or a pharmaceutically acceptable saltthereof may be administered at a dosage of at least 40 mg/day (in singleor divided doses). Additional embodiments for dosage amounts and rangesmay include (in single or divided doses):

-   -   (a) at least 48 mg/day    -   (b) at least 100 mg/day    -   (c) at least 120 mg/day    -   (d) at least 200 mg/day    -   (e) at least 240 mg/day    -   (f) at least 360 mg/day    -   (g) at least 480 mg/day    -   (h) from about 40 mg/day to about 480 mg/day    -   (i) from about 48 mg/day to about 240 mg/day    -   (j) from about 100 mg/day to about 300 mg/day    -   (k) from about 120 mg/day to about 300 mg/day    -   (l) from about 120 mg/day to about 240 mg/day    -   (m) from about 240 mg/day to about 480 mg/day    -   (n) about 48 mg/day    -   (o) about 120 mg/day    -   (p) about 240 mg/day    -   (q) about 360 mg/day    -   (r) about 480 mg/day

Although Compound (1) or a pharmaceutically acceptable salt thereof maybe administered in single or divided daily doses, once a dayadministration of the daily dose is preferred. As the skilled artisanwill appreciate, however, lower or higher doses than those recited abovemay be required. Specific dosage and treatment regimens for anyparticular patient will depend upon a variety of factors, including theage, body weight, general health status, sex, diet, time ofadministration, rate of excretion, drug combination, the severity andcourse of the infection, the patient's disposition to the infection andthe judgment of the treating physician. In general, the compound is mostdesirably administered at a concentration level that will generallyafford antivirally effective results without causing any harmful ordeleterious side effects.

The second component of the therapeutic combination, namelyinterferon-alfa, is comprised in a pharmaceutical composition.Typically, such compositions are injectable formulations comprisinginterferon-alfa and a pharmaceutically acceptable adjuvant or carrierand are well known in the art, including in a number of marketedinterferon-alfa formulations. See, e.g., the various marketedinterferon-alfa products and various patent and other literature relatedto interferon-alfa cited hereinabove.

The types of interferon-alfas that may be used in the combination are asoutlined hereinabove in the definitions section. In one preferredembodiment, the interferon alfa is a pegylated interferon alfa. In afurther embodiment, the interferon alfa is a pegylated interferonalfa-2a or pegylated interferon alfa-2b. In a particularly preferredembodiment, the interferon alfa is PEGASYS® or PEG-INTRON®.

When using known, marketed interferon alfa products, such products maybe administered at their labeled dosage levels indicated for interferonplus ribavirin combination therapy for the treatment of HCV infection.Of course, with the triple combination therapy of the present inventionit may be possible to use a lower dosage of interferon alfa, e.g.,significantly lower than is used the current standard interferon plusribavirin therapy, while delivering the same or better efficacy than thecurrent standard therapy with less side-effects usually associated withsuch therapy.

In one embodiment, the interferon alfa may be administered parenterallyone to three times per week, preferably once or twice a week. Withrespect to pegylated interferon alfas, these are typically administeredonce per week and the total weekly dose ranges, e.g., from about 0.5μg/kg/week to about 2 μg/kg/week in case of pegylated interferonalfa-2b, and with respect to pegylated interferon alfa-2a the dosage isindependent from the body weight of the host and is typically about 90to 200 μg/week, more preferably about 160 to about 200 μg/week. Incombination with ribavirin, a standard dosage of pegylated interferonalfa-2b is about 1.5 μg/kg/week and a standard dosage of pegylatedinterferon alfa-2a is about 180 μg/week, together with about 600-1200mg/day, in particular, 800-1200 mg/day of oral ribavirin.

According to further embodiments, the pegylated interferon alfa-2b maybe administered at dosages of:

-   -   (a) about 0.5 μg/kg/week to about 2 μg/kg/week;    -   (b) about 1 μg/kg/week to about 2 μg/kg/week;    -   (c) about 1.5 μg/kg/week to about 2 μg/kg/week;    -   (d) about 1.5 μg/kg/week

According to further embodiments, the pegylated interferon alfa-2a maybe administered at dosages of:

-   -   (a) about 90 to about 200 μg/week;    -   (b) about 160 to about 200 μg/week;    -   (b) about 180 μg/week

The third component of the therapeutic combination, namely ribavirin, iscomprised in a pharmaceutical composition. Typically, such compositionscomprise ribavirin and a pharmaceutically acceptable adjuvant or carrierand are well known in the art, including in a number of marketedribavirin formulations. Formulations comprising ribavirin are alsodisclosed, e.g., in U.S. Pat. No. 4,211,771.

The types of ribavirin that may be used in the combination are asoutlined hereinabove in the definitions section. In one preferredembodiment, the ribavirin is either REBETOL® or COPEGUS® and they may beadministered at their labeled dosage levels indicated for interferonplus ribavirin combination therapy for the treatment of HCV infection.Of course, with the triple combination therapy of the present inventionit may be possible to use a lower dosage of ribavirin, e.g., lower thanis used the current standard interferon plus ribavirin therapy, whiledelivering the same or better efficacy than the current standard therapywith less side-effects usually associated with such therapy.

According to various embodiments, the ribavirin may be administered atdosages of (in single or divided doses):

-   -   (a) between 400 mg/day to about 1200 mg/day;    -   (b) between about 800 mg/day to about 1200 mg/day;    -   (c) between about 1000 mg/day to about 1200 mg/day;    -   (d) about 1000 mg/day    -   (e) about 1200 mg/day    -   (f) between about 300 mg/day to about 800 mg/day    -   (g) between about 300 mg/day to about 700 mg/day    -   (h) between 500 mg/day to about 700 mg/day    -   (i) between 400 mg/day to about 600 mg/day    -   (j) about 400 mg/day    -   (k) about 600 mg/day    -   (l) about 800 mg/day

According to one embodiment, the ribavirin composition comprisesribavirin in a formulation suitable for dosing once a day, twice daily,thrice daily, four times a day, five times a day, or six times a day.For example, if a therapeutic combination comprises about 1000 mg/daydosage of ribavirin, and a dosing of five times a day is desired, thenthe therapeutic combination will comprise ribavirin in a formulation,e.g., a tablet, containing, e.g., about 200 mg of ribavirin.

With respect to the Compound (1) or a pharmaceutically acceptable saltthereof plus interferon alfa plus ribavirin triple combination therapyof the present invention, the present invention contemplates andincludes all combinations of the various preferred embodiments andsub-embodiments as set forth hereinabove.

For example, in one embodiment the present invention contemplates amethod of treating hepatitis C viral (HCV) infection or alleviating oneor more symptoms thereof in a patient comprising the step ofadministering to the patient a therapeutic combination comprising:

-   -   (a) Compound (1) or a pharmaceutically acceptable salt thereof        at a dosage between about 48 mg per day and about 480 mg per        day;    -   (b) pegylated interferon alfa-2a at a dosage of about 160 to        about 200 μg/week or pegylated interferon alfa-2b at a dosage of        about 0.5 μg/kg/week to about 2 μg/kg/week; and    -   (c) ribavirin at a dosage of between about 400 mg/day to about        1200 mg/day.

In another embodiment the present invention contemplates a method oftreating hepatitis C viral (HCV) infection or alleviating one or moresymptoms thereof in a patient comprising the step of administering tothe patient a therapeutic combination comprising:

-   -   (a) Compound (1) or a pharmaceutically acceptable salt thereof        at a dosage between about 48 mg per day and about 480 mg per        day;    -   (b) pegylated interferon alfa-2a at a dosage of about 180        μg/week; and    -   (c) ribavirin at a dosage of between about 1000 mg/day to about        1200 mg/day.

In another embodiment the present invention contemplates a method oftreating hepatitis C viral (HCV) infection or alleviating one or moresymptoms thereof in a patient comprising the step of administering tothe patient a therapeutic combination comprising:

-   -   (a) Compound (1) or a pharmaceutically acceptable salt thereof        at a dosage between about 48 mg per day and about 480 mg per        day;    -   (b) pegylated interferon alfa-2b at a dosage of about 1.5        μg/kg/week; and    -   (c) ribavirin at a dosage of about 800 mg/day.

Further embodiments include any of the above-mentioned embodiments, andwhere:

-   -   (a) the HCV infection is genotype 1 and the patient is a        treatment-naïve patient; or    -   (b) the HCV infection is genotype 1 and the patient is a        treatment-experienced patient who is non-responsive to a        combination therapy of interferon plus ribavirin.

Further embodiments include any of the above-mentioned embodiments, andwhere the Compound (1) or a pharmaceutically acceptable salt thereof isadministered once a day, the interferon alfa is administered once a weekand the ribavirin is administered twice a day.

According to a another embodiment, the therapeutic regimen of thepresent invention comprises administering to a patient for at leastabout 4 weeks, more preferably either at least about 12 weeks or atleast about 24 weeks:

(i) a therapeutically effective amount of Compound (1) or apharmaceutically acceptable salt thereof once a day;(ii) a therapeutically effective amount of interferon alfa once a week;and(iii) a therapeutically effective amount of ribavirin twice a day.

According to another embodiment, the present invention provides kits foruse in treating HCV infection in a patient. The kits of the presentinvention may comprise any of the therapeutic combinations of thepresent invention. The kits further comprise instructions for utilizingthe therapeutic combinations. The kits may be tailored to the needs ofclasses or types of patients or other clinically relevant factors suchas age, body weight, concomitant diseases/conditions, severity and stageof HCV infection, responsiveness or non-responsiveness to priortreatments, propensity for side effects, etc.

According to another embodiment, the present invention provides a kitcomprising:

(a) a first pharmaceutical composition comprising a Compound (1) or apharmaceutically acceptable salt thereof:(b) a second pharmaceutical composition comprising interferon alfa;(c) a third pharmaceutical composition comprising ribavirin; and.(d) instructions for utilizing the above compositions.

Additionally, surprising results have been seen in the suppression ofHCV viral resistance during the combination therapy treatmentcontemplated by the present invention. Dosing of Compound (1) (sodiumsalt) as a monotherapy resulted in a rapid viral load rebound in thefirst 14 days during treatment in a majority of the patients from alldose groups of treatment-naïve patients in whom viral load reductionswere observed. In contrast, among the 19 treatment experienced patientswho received Compound (1) (sodium salt) once daily (qd) doses of 48 mg(n=6), 120 mg (n=7), or 240 mg (n=6) in a combination with pegylatedinterferon alfa 2a and ribavirin (PegIFN/RBV) for 28 days, virologicrebound during the 28 day treatment was only observed in 2/6 patients inthe 48 mg dose group, and in 1/7 patients in the 120 mg dose group.Notably, no rebound was seen during the first 28 days in thetreatment-experienced dose group that received Compound (1) (sodiumsalt) 240 mg qd treatment in combination with PegIFN/RBV.

Accordingly, in an additional embodiment, there is limited or noemergence of viral resistance during the combination therapy of thepresent invention. In a more specific embodiment, there is limited or noemergence of HCV variants that encode HCV NS3 protease amino acidsubstitutions at one or more of R155 and/or D168 and/or A156 during thecombination therapy of the present invention.

Further embodiments include any of the above-mentioned embodiments, andwhere either:

-   -   (a) the HCV infection is genotype 1a and the patient is a        treatment-naïve patient; or    -   (b) the HCV infection is genotype 1a and the patient is a        treatment-experienced patient who is non-responsive to a        combination therapy of interferon plus ribavirin;        and wherein there is limited or no emergence of variants that        encode substitutions at NS3 protease amino acid R155 during the        combination therapy of the present invention.

Further embodiments include any of the above-mentioned embodiments, andwhere either:

-   -   (a) the HCV infection is genotype 1b and the patient is a        treatment-naïve patient; or    -   (b) the HCV infection is genotype 1b and the patient is a        treatment-experienced patient who is non-responsive to a        combination therapy of interferon plus ribavirin;        and wherein there is limited or no emergence of the variants        that encode substitutions at NS3 protease amino acid D168 during        the combination therapy of the present invention.

EXAMPLES I. Methods for Preparing Compound (1)

Methods for preparing amorphous Compound (1) can be found in U.S. Pat.Nos. 6,323,180, 7,514,557 and 7,585,845, which are herein incorporatedby reference. The following Examples 1 to 5 provide methods forpreparing additional forms of Compound (1) that may be used in thepresent invention.

Example 1 Preparation of Type A Crystalline Form of Compound (1)

Amorphous Compound (1) (Batch 7, 13.80 g) was added to a 1000 ml threeneck flask. Absolute ethanol (248.9 g) was added to the flask. Whilestirring, the contents of the flask were heated at 60 degrees C./hr to˜74 degrees C. (Solids do not dissolve at 74 degrees C.). Water (257.4g) was then added linearly over 4 hr to the resulting slurry whilestirring and maintaining the temperature at 74 degrees C. After thewater addition was complete, the temperature was reduced linearly toambient temperature at 8 degrees C./hr and then held at ambienttemperature for 6 hrs while stirring. The resulting solids werecollected by filtration and washed with 50 ml of 1/1 (w/w) EtOH/Water.The wet solids were dried on the funnel for 30 minutes by sucking N2through the cake. (XRPD analysis on this sample indicates that thepattern is similar to the EtOH solvate). The solids were then dried at65-70 degrees C. under vacuum (P=25 in Hg) and a nitrogen bleed for 1.5hr. The resulting solids (12.6 g, 95.5% corrected yield) were confirmedby XRPD as being Type A Compound (1).

Example 2 Preparation of the Sodium Salt of Compound (1) Method 1

2.1 g of amorphous sodium salt of Compound (1) and 8.90 g of acetone wasadded to a vial and stirred at ambient temperature for 3 hr. The slurrywas filtered off mother liquors and the resulting solids were dried for20 minutes under nitrogen flow for 20 minutes. 1.51 g of crystallinesodium salt of Compound (1) as solids was collected.

Example 3 Preparation of the Sodium Salt of Compound (1) Method 2

15.6 g of Type A of Compound (1), 175 ml of acetone and 3.6 ml of waterwas added to a 250 ml reactor and heated to 53 degrees C. to dissolvethe solids. 900 ul of 10.0 N NaOH was added to reactor and the solutionwas seeded with Type A. The seeded solution was stirred at 53 degrees C.for 10 minutes. A second 900 ul portion of 10.0 N NaOH was added and thesystem was stirred at 53 degrees C. for 30 minutes over which a slurrydeveloped. The slurry was cooled to 19 degrees C. at a cooling rate of15 degrees C. per hour and held overnight at 19 degrees C. The finalresulting slurry was filtered and the wet solids were washed with 15 mlof acetone. Dried solids for 1 hr at 52 degrees C. under vacuum with anitrogen flow and then exposed the solids to lab air for one hour.Collected 12.1 g of Compound (1) crystalline sodium salt solids.

Example 4 Preparation of the Sodium Salt of Compound (1) Method 3

25.4 Kg of amorphous Compound (1), 228 L of THF and 11.1 Kg of 10 wt %NaOH (aq) was added to a reactor. The components were mixed at 25degrees C. to dissolve all solids. The resulting solution was filteredand the reactor and filter was washed with 23 L of THF. 180 L of solventwas removed using atmospheric distillation at 65 degrees C. 195 L ofMIBK was added and 166 L of solvent was removed by vacuum distillationat ±44 degrees C. 161 L of MIBK and 0.41 Kg of water was added back tothe reactor and the contents were heated to 70 degrees C. 255 g ofCompound (1) sodium salt seeds were added at 70 degrees C. and 1.42 L ofwater was added over 1.5 hours. After the water addition the slurry washeld at 70 degrees C. for 45 minutes and then cooled to 45 degrees C.over 1 hr. The resulting slurried was filtered and washed with 64 L ofMIBK containing ˜0.8 weight % water. The wet cake was dried at 55degrees C. to give ˜25 Kg of crystalline sodium salt of Compound (1).

Example 5 Preparation of the Sodium Salt of Compound (1) Method 4

2.00 g of amorphous Compound (1), 9.96 g of THF and 0.11 g of water wasadded to a reactor and stirred at ambient temperature to dissolvesolids. 0.820 ml of 21 weight % NaOET in ethanol was added drop-wisewhile stirring the solution to get solution A. 15.9 g of n-BuAc and 160ul of water was added to a second reactor and heated to 65 degrees C.(solution B). 2.56 g of Solution A was added to Solution B at 65 degreesC. and the resulting mixture was seeded with 40 mg of Compound (1)sodium salt seeds. The seeded mixture was aged at 65 degrees C. for 45minutes. 2.56 g of Solution B was added to Solution A and aged for 45minutes in four separate intervals. After the final addition and aging,the slurry was cooled to 50 degrees C. over 1 hour and filtered. The wetcake was washed with 6 ml of n-BuAc containing 0.5 weight % water. Thefinal solids were dried at 50 degrees C. under vacuum using a nitrogenpurge. Compound (1) crystalline sodium salt solids were collected.

II. Clinical Results

For the clinical trials described below, the drug product administeredwas an oral solution of Compound (1) sodium salt. The Compound (1)sodium salt was provided to the clinical site(s) as a powder forpreparing an oral solution using a co-supplied solvent. The solvent wasalso used as the placebo.

Example 6 Clinical Study with Treatment-Naïve Patients

Safety and antiviral activity of Compound (1) sodium salt, a new HCV NS3protease inhibitor, in treatment-naive patients with chronic hepatitis Cgenotype-1 infection given as monotherapy and in combination withPeginterferon alfa 2a (P) and Ribavirin (R)

Background: Compound (1) sodium salt is a HCV NS3 protease inhibitor(EC50 of 3-6 nM). A multiple rising dose study evaluated safety andantiviral activity in treatment-naive patients (pts) with chronic HCVgenotype-1 infection as monotherapy for 14 days followed by triplecombination therapy with P+R for an additional 14 days.

Methods: 34 patients (France, Germany, Spain, USA) with a Metavirfibrosis score of 0-3 and no prior therapy with any interferon or R wererandomized (2 placebo:6 or 7 active) to 4 dose groups of once-daily (qd)Compound (1) sodium salt: 20 mg (n=8), 48 mg (n=9), 120 mg (n=9), or 240mg (n=8). Compound (1) sodium salt was given as monotherapy for 14 days.Pts with <1 log₁₀ decrease in Day 10 viral load (VL) had Compound (1)sodium salt discontinued after Day 14. Pts with a ≧1 log 10 decrease inDay 10 VL continued Compound (1) sodium salt on Day 15 and added P(180μg/week)+R(weight based) for triple combination therapy through Day 28.The primary endpoint was ≧2 log 10 VL reduction at any time to Day 14.Plasma HCV-RNA levels were measured with Roche COBAS® TaqMan® assay(LLOQ 25 IU/m L). After day 28, at the discretion of the investigator,patients could continue to receive standard of care treatment, i.e.,P+R.

Results: 33 pts were white, 1 was Asian, 27 were male, meanage=48.9±11.1 years, mean body weight 79.1±17.5 kg, and median (range)baseline VL was 6.8 (4.7-7.7) log 10. There were no significantdemographic differences between dose groups. Compound (1) sodium saltwas well tolerated. No pts discontinued treatment during monotherapy dueto adverse events (AEs). AEs observed were typical for P+R. One seriousAE, asthenia, occurred in the 20 mg dose cohort 6 days after initiatingP+R. Rapid decline of VL was observed in all pts with maximal declinetypically 2-4 days after starting Compound (1) sodium salt. With theexception of 1 pt in the 20 mg cohort, all pts on Compound (1) sodiumsalt achieved >2 log 10 VL decline during the monotherapy period. Median(range) maximal reductions in VL during 14 day monotherapy for the 20mg, 48 mg, 120 mg, and 240 mg groups were 3.0 (1.5-3.9), 3.6 (3.1-3.8),3.7 (3.3-4.1), and 4.2 (3.6-4.8) log 10 IU/ml, respectively. Nosignificant change in VL was observed with placebo. VL rebound duringtreatment was seen in the first 14 days of monotherapy in a majority ofpatients from all dose groups.

Conclusion: Compound (1) sodium salt as monotherapy for 14 days followedby combination with P+R for additional 14 days was well tolerated, andinduced a strong and rapid antiviral response among treatment naïvepatients.

FIG. 1 depicts the average change in HCV viral load in the four patientdose groups of this study, i.e., treatment-naïve patients with chronicHCV genotype-1 infection treated with Compound (1) sodium salt asmonotherapy for 14 days, followed by combination therapy with Compound(1) sodium salt, pegylated interferon alfa-2a and ribavirin for anadditional 14 days. In the figure, the change in viral load is in IU/mllog 10 units and the four patient dose groups are labeled as follows:

-   -   “20 naiv av”=average viral load change for 20 mg dose group    -   “48 naiv av”=average viral load change for 48 mg dose group    -   “120 naiv av”=average viral load change for 120 mg dose group    -   “240 naiv av”=average viral load change for 240 mg dose group        Emergence of Variants During Treatment-Naïve Patient Therapy

Population sequencing of the NS3/4A protease at baseline and reboundduring treatment revealed selection of variants that confer in vitroresistance to Compound (1) sodium salt. There were seen notable changesat key residues within the NS3 protease domain relative to subtypereference (sub-genotype 1a: AF009606 or sub-genotype 1b: AJ238799)during therapy in various dose groups. See Tables 1 to 4 below fordetails. In all tables, Gt represents the HCV subgenotype and Day 1represents baseline sequence and the indicated amino acid substitutionsare naturally occurring NS3 polymorphisms at key residues. Additionalchanges, that may encode or be associated with drug-resistance, arenoted at different days during treatment.

TABLE 1 Predominant variants (relative to subtype reference) at keyresidues in the population-based amino acid sequences from thetreatment-naive patients in the 20 mg dose group Day of treatmentPatient Gt 1 6 14 28 56 168 20N1 1b I71V, I71V, Q86P I71V, ND ND ND Q86PQ86P 20N2 1b none BLD D168V R155[K, Q, R], R155[K, Q, R], BLD D168[D, V]D168[D, V] 20N3* 1b Q86A, Q86A, Q86A, BLD BLD BLD V/I170T V/I170TV/I170T 20N4 1a none V/I170[I, T] R155K R155K R155K BLD 20N5 1b Q80L,Q80L, Q80L, BLD BLD BLD D168[G, A] D168[E, D] 20N6 1a none none none NDND ND 20N7 1a none R155K R155K R155K R155K R155K 20N8 1a none R155[R, K]R155K R155K R155K BLD none: no substitutions at the selected positionswere observed BLD: Below limit of detection for PCR amplification ND:Not determined *Encodes V/I170T baseline polymorphism.

TABLE 2 Predominant variants (relative to subtype reference) at keyresidues in the population-based amino acid sequences from thetreatment-naïve patients in the 48 mg dose group. Day of treatmentPatient Gt 1 14 28 48N1 1a T54[T, S] T54[T, S] ND 48N2 1b I71V, Q86PI71V, Q86P ND V170[E, V] 48N3 1b P89S P89S, D168V P89S, D168V 48N4 1bI71V I71V, D168V T54[T, A], I71V, D168V 48N5 1a none R155K R155K 48N6 1bnone D168[T, I, A, V] D168[T, I, A, V] 48N7 1b none BLD BLD 48N8 1a Q80KQ80K, R155K BLD 48N9 1b I71V, I71V, Q80[Q, R] ND Q80[Q, R] R155[R, W],D168[D, V] None: no substitutions at the selected positions wereobserved BLD: Below limit of detection for sequencing ND: Not determined

TABLE 3 Predominant variants (relative to subtype reference) at keyresidues in the population-based amino acid sequences from thetreatment-naive patients in the 120 mg dose group Day of treatmentPatient Gt 1 14 28 84 120N1 1a Q80K Q80K ND ND 120N2 1 D168D/E D168D/END ND 120N3 1a none R155K BLD BLD 120N4 1a Q80K BLD ND ND 120N5 1b noneD168V BLD BLD 120N6 1b P89T P89T, P89T, P89T, D168V D168V D168V 120N7 1anone R155K BLD BLD 120N8 1b none D168V D168V BLD 120N9 1b Q86P Q86P,Q86P, Q86P, A156[T, A], D168[D, I, A, R155K D168[D, V] V] none: nosubstitutions at the selected positions were observed BLD: Below limitof detection for sequencing ND: Not determined

TABLE 4 Predominant variants (relative to subtype reference) at keyresidues in the population-based amino acid sequences from thetreatment-naive patients in the 240 mg dose group Day of TreatmentPatient Gt 1 14 28 84 240N1 1b Q8OL, Q86P Q80L, Q86P ND ND 240N2 1b nonenone ND ND 240N3 1a V36[L, V], V36L, ND V36L, Q80K Q80K, Q80K, R155KR155K 240N4 1b none BLD BLD BLD 240N5 1b P89A P89A, P89A, BLD D168VD168V 240N6 1 none D168V D168V BLD 240N7 1b none D168V BLD BLD 240N8 1aQ89P BLD BLD BLD none: no substitutions at the selected positions wereobserved BLD: Below limit of detection for PCR amplification andsequencing ND: Not determined

Example 7 Clinical Study with Treatment-Experienced Patients

Safety and antiviral activity of Compound (1) sodium salt, a new HCV NS3protease inhibitor, in combination therapy with Peginterferon alfa 2a(P) and Ribavirin (R) for 28 days in P+R treatment-experienced patientswith chronic hepatitis C genotype-1 infection.

Background: Compound (1) sodium salt is a HCV NS3 protease inhibitor(EC50 of 3-6 nM). A multiple rising dose study evaluated the safety andantiviral activity in P+R treatment-experienced patients (pts) withchronic hepatitis C genotype-1 infection for 28 days as combinationtherapy with P+R.

Methods: 19 pts (France, Germany, Spain, USA) with a Metavir fibrosisscore of 0-3, who experienced previous virologic failure with P+Rcombination therapy, were assigned to receive Compound (1) sodium saltonce-daily (qd) doses of 48 mg (n=6), 120 mg (n=7), or 240 mg (n=6) incombination with P (180 μg/wk)+R (weight based) for 28 days. Allpatients were monitored for safety and tolerability of study drugs. Theprimary endpoint was a ≧2 log 10 reduction in HCV viral load (VL) frombaseline at any time up to Day 28. Plasma HCV-RNA levels were measuredusing the Roche COBAS® TaqMan® assay (LLOQ 25 IU/mL). The treatmentexperienced patients in this study included P+R null responders andpartial responders. After day 28, at the discretion of the investigator,patients could continue to receive standard of care treatment, i.e.,P+R.

Results: 19 pts were white, 11 were male, mean age was 48±9 years, meanbody weight was 81±15 kg, and median (range) baseline VL was 6.9(5.9-7.4) log 10. There were no significant demographic differencesbetween dose groups. Compound (1) sodium salt was well tolerated and noserious or severe adverse events (AEs) were observed among pts in thisstudy. AEs were typical for P+R. One subject discontinued treatment dueto an AE (anxiety). A rapid, dose-related decline of VL was observed inall pts. All pts treated with Compound (1) sodium salt+P+R achieved >2log 10 VL decline with triple combination therapy. Median (range)maximal decline in VL during 28 day combination therapy for 48 mg, 120mg, and 240 mg dose cohorts was 4.8 (3.4-5.9), 5.2 (3.9-6.0), and 5.3(4.8-6.1) log 10 IU/ml, respectively. Virologic rebound during treatmentwas observed during the first 28 days of Compound (1) sodium salt+P+Rdosing in 2/6 pts in the 48 mg and in 1/7 pts in the 120 mg dose groups.In these patients, population sequencing of the NS3/4A protease atbaseline and at viral rebound during treatment revealed selection ofvariants in the NS3 protease domain shown to confer in vitro resistanceto Compound (1) sodium salt. One patient in the 120 mg dose group didnot display virologic rebound, but plateaued with ˜500 copies IU/ml VLby day 28 and encoded a R155K mutant; this viral load sample was belowthe lower limit of detection in our phenotypic resistance assays.

No rebound during the 28-day BI 201335 treatment was seen in the 240 mgqd dose cohort: ⅚ pts had VL<25 IU/mL at Day 28. The sixth pt had a 4.7log 10 decline in VL from baseline on Day 28 and VL was <25 IU/ml atnext visit, Day 42.

Conclusion: Compound (1) sodium salt given once-daily in combinationtherapy with P+R for 28 days was well tolerated, and induced a strongand rapid antiviral response among treatment experienced patients.

FIG. 2 depicts the average change in HCV viral load in three patientdose groups for this study, i.e., treatment-experienced patients withchronic HCV genotype-1 infection treated with Compound (1) sodium salt,pegylated interferon alfa-2a and ribavirin as combination therapy for 28days. In the figure, the change in viral load is in IU/ml log 10 unitsand the four patient dose groups are labeled as follows:

-   -   “48 NR av”=average viral load change for 48 mg dose group    -   “120 NR av”=average viral load change for 120 mg dose group    -   “240 NR av”=average viral load change for 240 mg dose group

Certain parameters relating to the virologic response (reduction inviral load) in the patients in this study are shown in the followingtable (where “D”=day, and “QD”=once per day; “N”=# of patients; “BelowLimit of Quantification” means less than 25 IU per ml of serum).

1220.2 Virologic Response in Treatment-experienced Patients 48 mg 120 mg240 mg QD QD QD N = 6 N = 7 N = 6 Maximal decline 4.4 4.5 4.8 D 1-14(×log₁₀) (3.4-4.9) (3.6-5.2) (4.2-5.1) Mean decline at 3.8 4.0 4.2 D 6(×log₁₀) Mean Decline at 4.1 4.3 4.8 D 14 (×log₁₀) Below Limit of 3 4 5Quantification (50%) (57%) (83%) at D 28

Emergence of Variants During Treatment-Experienced Patient Therapy

There are notable changes at key residues within the NS3 protease domainrelative to subtype reference (sub-genotype 1a: AF009606 or sub-genotype1b: AJ238799) during therapy in various dose groups. See Tables 1 to 4below for details. In all tables, Gt represents the HCV subgenotype andDay 1 represents baseline sequence and the indicated amino acidsubstitutions are naturally occurring NS3 polymorphisms at key residues.Additional changes, that may encode or be associated withdrug-resistance, are noted at different days during treatment.

TABLE 5 Predominant variants (relative to subtype reference) at keyresidues in the population-based amino acid sequences from the 48 mgtreatment-experienced dose group. Day of treatment Patient Gt 1 14 28168 48TE1 1a Q80K Q80K Q80K, R155K Q80K, R155K 48TE2 1a Q80L BLD BLD BLD48TE4 1a Q80R BLD BLD BLD 48TE3 1b none D168V D168V R155R/K/Q 48TE5 1bQ86P BLD BLD BLD 48TE6 1b Q86P BLD BLD Q86P, R155K none: nosubstitutions at the selected positions were observed BLD: Below limitof detection for PCR amplification and sequencing

TABLE 6 Predominant variants (relative to subtype reference) at keyresidues in the population-based amino acid sequences from the 120 mgtreatment-experienced dose group. Day of Treatment Patient Gt 1 14 28120TE1 1a Q80K Q80K, R155K Q80K, R155K 120TE2 1a Q89Y BLD BLD 120TE3 1bQ86P, P89S BLD BLD 120TE4 1b Q86A BLD BLD 120TE5 1b P89[P, S] BLD BLD120TE6 1a none BLD BLD 120TE7 1a none R155K* R155K* None: nosubstitutions at the selected positions were observed BLD: Below limitof detection for PCR amplification and sequencing *VL plateau at <1000IU/ml at these time points.

TABLE 7 Predominant variants (relative to subtype reference) at keyresidues in the population-based amino acid sequences from the 240 mgtreatment-experienced dose group. Day of Treatment Patient Gt 1 28240TE1 1a none BLD 240TE2 1a none BLD 240TE3 1a none BLD 240TE4 1b noneBLD 240TE5 1b Q86P BLD 240TE6 1a none BLD None: no substitutions at theselected positions were observed BLD: Below limit of detection for PCRamplification and sequencing

Overall Assessment

These results demonstrate a strong and very rapid antiviral affect intreatment-naïve patients treated with Compound (1) sodium salt givenonce-daily as monotherapy for 14 days, which is followed by anti-viralresistance formation and increase in viral load after 5-6 days ofmonotherapy. Upon initiation of the triple combination therapy of thepresent invention at day 14 (Compound (1) sodium salt, with pegylatedinterferon alfa 2a and ribavirin), however, viral load progressivelydecreased demonstrating the antiviral effectiveness of the triplecombination therapy of the present invention in treatment-naïvepatients. See FIG. 1.

When Compound (1) sodium salt was given once-daily totreatment-experienced patients (interferon plus ribavirin nullresponders and partial responders) in combination with pegylatedinterferon alfa 2a and ribavirin for 28 days, the results demonstratethe same strong and very rapid antiviral response but with reducedresistance formation. See FIG. 2. One would have expected in suchtreatment-experienced, non-responder patients that a similar viralresistance would have occurred after day 5 as these patients wereselected for being non-responsive to pegylated interferon plus ribavirintherapy. However, a continuous viral suppression occurred.

In essence, it has been shown that the triple combination therapy of thepresent invention can effectively reduce the viral load oftreatment-naïve and treatment-experienced patients with chronicgenotype-1 hepatitis C viral infection and, at least in some patients,keep it at less than detectable level, that is defined as below 50International Units per ml serum of a patient as measured byquantitative, multi-cycle reverse transcriptase PCR methodologyaccording to the WHO international standard. In preferred embodiments,the triple combination therapy of the present invention can effectivelyreduce the viral load in HCV genotype 1-infected patients to below 10International Units per ml serum.

Definitely surprising results include:

(1) no early resistance formation (e.g., variants encoding NS3 aminoacid substitutions at R155 and/or D168) in treatment-experiencedpatients who are treated with Compound (1) sodium salt in combinationwith pegylated interferon alfa 2a and ribavirin at standard doses (FIG.2) as compared to the effect of the same dose of Compound (1) sodiumsalt alone in treatment-naïve patients who experienced viral resistance(see FIG. 1, first 14 days). One would have expected in treatmentexperienced patients that a similar viral resistance would have occurredafter day 5 even with the addition of pegylated interferon alfa 2a andribavirin as these patients were selected for being non-responsive toprevious pegylated interferon plus ribavirin combination therapy; and(2) that a dose of only 120 mg QD (once daily) in combination withpegylated interferon and ribavirin induced a viral depletion to belowthe level of quantification (defined as less than 25 IU per ml of serum)in more than 50% of the treatment-experienced patients who have beenpreviously non-responsive to pegylated interferon plus ribavirin therapy(see table).

Example 8 Methods for Identifying HCV NS3 Variants Viral RNA Extractionand PCR Amplification

Viral RNA was isolated from plasma of HCV-infected subjects and a DNAfragment of 2.4 kbp containing the complete NS3-NS4A region was firstsynthesized using SUPERSCRIPT™ III one step RT-PCR System (Invitrogen)and two gene-specific primers spanning positions 3276 in NS2 and 5650 inNS4B. After purification of the first PCR product, two differentsecond-round, semi-nested PCR products of either 2.3 or 0.7 kbp(spanning the entire NS3/NS4A or only the NS3 protease domain,respectively) were then generated using KOD Hot Start DNA polymerase(Novagen). The limit of detection of the RT-PCR amplification methodrestricted the analysis to patient samples with VL above 1000 IU/ml.

Sequence Analysis

The 2.3 kbp DNA product was then used for direct population-basedsequencing of the entire N53-NS4A 2055 nt region using BIG DYE®Terminator V3.1 (Applied Biosystems) and an ABI PRISM® 3130XL GeneticAnalyzer (Applied Biosystems). Sequences were obtained from 10 primersto achieve at least 90% double strand coverage for the NS3-NS4A region.The resulting nucleotide sequences were analyzed with SEQSCAPE® v2.5(Applied Biosystems).

The 0.7 kbp DNA fragment was used to generate the clonal-based(ZEROBLUNT® TOPO® Cloning Kit, Invitrogen) sequences of the 543 nt NS3protease region covering the first 181 aa of NS3; for each sample 96clones were picked and sequenced using universal primers with ABI PRISM®BIG DYE™ Terminator Cycle PCR Sequencing. Two single pass sequences wereperformed for each clone resulting in 90-100% double strand coverage ofthe 543 nt region analyzed with MUTATION SURVEYOR™ v3.0 (SoftgeneticsLLC). For each patient sample, the clones with low quality sequence orcontaining deletions, insertions or stop codons were not included forfurther analysis such that the number of clones analyzed varied from 74to 89 (with a median and average of 80 clones).

The resulting sequences were compared to reference sequences accordingto their respective subtypes which were previously determined during thetrial screening phase with the TRUEGENE™ HCV 5′NC genotyping assay.AF009606 served as the reference for subtype 1a and AJ238799 for subtype1b. Particular attention was focused on mutations that resulted in aminoacid substitutions at 15 positions in the NS3 protease domain. Thesepositions were all previously reported as potentially conferringresistance to this class of compounds. These positions are: 36, 41, 54,71, 80, 86, 89, 109, 111, 155, 156, 168, 170, 176 and 178 (References:[1.] Tong X, Bogen S, Chase R, Girijavallabhan V, Guo Z, Njoroge F G,Prongay A, Saksena A, Skelton A, Xia E, Ralston R. Characterization ofresistance mutations against HCV ketoamide protease inhibitors.Antiviral Res. 2008 March 77(3):177-185. [2.] Lagace L, Marquis M,Bousquet C, Do F, Gingras R, Lamarre D, Lamarre L, Maurice R, Pause A,Pellerin C, Spickler C, Thieault D, and Kukolj G. BILN 2061 and beyond:pre-clinical evaluation of HCV subgenomic replicon resistance to a NS3protease inhibitor. In Framing the Knowledge of Therapeutics for ViralHepatitis. R F Schinazi and E R Schiff (eds). P263-278 IHL press, 2006[3.] Koev G, Kati W. The emerging field of drug resistance. ExpertOpinion Invest Drugs 17(3), 303-319, 2008 (P08-03895)

Drug Sensitivity Assays

A bicistronic HCV replicon shuttle vector (pIT2) comprising a luciferasereporter gene and an adapted Con-1 NS3/NS5B region was modified tocreate two unique restriction sites (Mlu I and Spe I) at NS3 codons 11and 225 which enabled the insertion of compatible NS3 amplicons isolatedfrom HCV-infected patient plasma samples. The first-round PCR productsynthesized from the patient plasma-purified RNA (also used forgenerating fragments for population- and clonal-based sequencing) wasused to amplify a 0.65 kbp fragment with primer pairs that respectivelycontain the unique MluI and SpeI restriction sites for insertion intothe shuttle vector. Amplicons were ligated in the pIT2 shuttle vectorand reconstituted plasmid DNA was used to generate HCV subgenomicreplicon RNA transcripts (T7 RIBOMAX™ kit, Promega). The in vitrotranscribed RNA was transiently transfected by electroporation inHuh-7.5 cells which were then seeded in 96 well plates for 24 hours andtreated with a range of Compound (1) sodium salt (or IFN-α)concentrations for a period of 72 hours. At the end of the incubation,luciferase activity was measured with the BRIGHT-GLO™ substrate as theluminescence quantified (CPS) in each well of the culture platereflected the amount of HCV RNA replication. The level of inhibition (%inhibition) in each well containing inhibitor was calculated with thefollowing equation: % inhibition=100−[100×CPS(inhibitor)/CPS(control)].The concentration giving 50% inhibition of HCV RNA replication (EC₅₀)was determined by the non-linear regression routine NLIN procedure ofSAS. The EC₅₀ of mutant NS3 were compared to the baseline EC₅₀ and usedto generate fold-change values.

The predominant genotype 1a resistance mutations in on-treatment viralrebound samples encoded an R155K substitution, and other minor variantswere detected by clonal sequence analysis at this position. R155Kvariants conferred reductions in sensitivity to BI 201335 with the rangeof EC50 values of 1.8-6.5 μM. The genotype 1b viruses mainly encodedchanges at D168, with valine as a predominant substitution, and otherminor variants detected by sensitive clonal sequencing. EC₅₀ values forD168 variants ranged 3.6-15 μM. This profile may be attributable, inpart, to a different mutational barrier to resistance at the R155 codonin genotype 1a (a single nucleotide transition changes the codon to alysine) versus genotype 1b (that requires two nucleotide substitutionsto encode a change to lysine.

1. A method of treating hepatitis C viral (HCV) infection or alleviating one or more symptoms thereof in a patient comprising the step of administering to the patient a therapeutic combination comprising: (a) a compound of the following formula (1) or a pharmaceutically acceptable salt thereof:

(b) interferon alfa; and (c) ribavirin.
 2. The method according to claim 1, wherein the HCV infection is genotype
 1. 3. The method according to claim 1, wherein said patient is a treatment-naive patient.
 4. The method according to claim 1, wherein said patient is non-responsive to a combination therapy using ribavirin and an interferon alfa.
 5. The method according to claim 1, wherein the HCV-RNA levels of said patient are reduced to a less than detectable level as a result of the treatment.
 6. The method according to claim 1, wherein said therapeutic combination is administered for at least 4 weeks.
 7. The method according to claim 1, wherein said therapeutic combination is administered for at least 12 weeks.
 8. The method according to claim 1, wherein said therapeutic combination is administered for at least 24 weeks.
 9. The method according to claim 1, wherein compound (1) or a pharmaceutically acceptable salt thereof is administered at a dosage of at least 40 mg per day.
 10. The method according to claim 1, wherein compound (1) or a pharmaceutically acceptable salt thereof is administered at a dosage between about 40 mg per day and about 300 mg per day.
 11. The method according to claim 1, wherein compound (1) or a pharmaceutically acceptable salt thereof is administered at a dosage between about 48 mg per day and about 240 mg per day.
 12. The method according to claim 1, wherein compound (1) is administered in the form of its sodium salt.
 13. The method according to claim 1, wherein said ribavirin is administered at a dosage between about 400 mg/day and about 1200 mg/day.
 14. The method according to claim 1, wherein said ribavirin is administered at a dosage between about 800 mg/day and about 1200 mg/day.
 15. The method according to claim 1, wherein said interferon alfa is administered once a week.
 16. The method according to claim 1, wherein said interferon alfa is a pegylated interferon alfa.
 17. The method according to claim 16, wherein said pegylated interferon alfa is pegylated interferon alfa-2a or pegylated interferon alfa-2b.
 18. The method according to claim 16, wherein the pegylated interferon alfa is pegylated interferon alfa-2b administered at a dosage of about 0.5 μg/kg/week to about 2 μg/kg/week.
 19. The method according to claim 16, wherein the pegylated interferon alfa is pegylated interferon alfa-2b administered at a dosage of about 1 μg/kg/week to about 2 μg/kg/week.
 20. The method according to claim 16, wherein the pegylated interferon alfa is pegylated interferon alfa-2b administered at a dosage of about 1.5 μg/kg/week.
 21. The method according to claim 16, wherein the pegylated interferon alfa is pegylated interferon alfa-2a administered at a dosage of about 90 to 200 μg/week.
 22. The method according to claim 16, wherein the pegylated interferon alfa is pegylated interferon alfa-2a administered at a dosage of about 180 μg/week.
 23. The method according to claim 1, wherein the HCV infection is genotype 1, the patient is non-responsive to a combination therapy using ribavirin and an interferon, the compound (1) or a pharmaceutically acceptable salt thereof is administered at a dosage between about 48 mg per day and about 240 mg per day and wherein said interferon alfa is pegylated interferon alfa-2a or pegylated interferon alfa-2b.
 24. The method according to claim 23, wherein compound (1) is administered in the form of its sodium salt.
 25. The method according to claim 1, comprising administering to the patient compound (1) or a pharmaceutically acceptable salt thereof once a day; ribavirin twice a day; and interferon alfa once a week.
 26. The method according to claim 23, comprising administering to the patient compound (1) or a pharmaceutically acceptable salt thereof once a day; ribavirin twice a day; and interferon alfa once a week.
 27. The method according to claim 1, wherein there is limited or no emergence of HCV variants that encode HCV NS3 protease amino acid substitutions at one or more of: R155, D168, or A156.
 28. A kit comprising: (a) a first pharmaceutical composition comprising a compound of the following formula (1) or a pharmaceutically acceptable salt thereof:

(b) a second pharmaceutical composition comprising interferon alfa; and (c) a third pharmaceutical composition comprising ribavirin. 